Anticancer agents based on regulation of protein prenylation

ABSTRACT

Oncoproteins such as Ras and RhoB are known to induce cell division in an unrestrained manner when such proteins are localized at the inner surface of a cancer cell membrane. The localization is effected by the prenylation reaction, whereby a hydrophobic group (e.g. a farnesyl group) is attached to the protein in the presence of an enzyme (e.g. farnesyl protein transferase). Deactivation of the prenylation enzyme through covalent modification can therefore ultimately result in the mitigation and/or cessation of cancer cell growth. Various prenylation inhibitors having the necessary structural groups to bond covalently, or essentially irreversibly, to the prenylation enzyme include carbonyl or thiocarbonyl compounds (or masked versions of these compounds) and alpha oxo-epoxides bonded to a hydrophobic, substrate-mimicking group. The carbonyl or thiocarbonyl compounds also contain a nucleofugal atom or group to enhance the tendency to form covalent bonds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/275,662, filed Jan. 23, 2006, now allowed, which is a division ofapplication Ser. No. 09/983,232, filed Oct. 23, 2001, now U.S. Pat. No.7,019,031, which claims priority to Provisional Application Ser. No.60/241,955, filed Oct. 23, 2000. The disclosure of each of theseapplications is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the prevention and/or treatment ofcancer by inhibiting enzyme-catalyzed prenylation reactions that allowlocalization of Ras, RhoB, and other proteins at the inner surfaces ofcancer cell membranes and other intracellular locations, thereby causingunrestrained cell division.

BACKGROUND OF THE INVENTION

Approximately 25% of all human cancers result from a mutant gene thatencodes a mutant form of the protein known as Ras. In cancer cells, Rasactivates the cells to divide in an unrestrained manner. To induce celldivision, Ras must be localized at the inner surface of the cancer cellmembrane. This membrane localization of Ras is effected by attachment ofa hydrophobic group, typically the farnesyl group, but possibly therelated geranylgeranyl group. In either case, the group becomes attachedto Ras enzymatically in a process known as prenylation. Thus,interference with prenylation of Ras has the potential to prevent Raslocalization at the inner surface of the cancer cell membrane, resultingin the cessation of unrestrained cell division and/or reversion of thecancer cell to a normal phenotype.

The enzyme that attaches the farnesyl group to Ras, RhoB, and otherproteins to facilitate the proper localization of these proteins in thecell is farnesyl protein transferase, also known as proteinfarnesyltransferase (referred to here as FTase). The farnesyl groupbecomes attached to Ras, RhoB, and other proteins by reaction withfarnesyl diphosphate, also known as farnesyl pyrophosphate (referred tohere as FPP). In other words, FTase catalyzes the reaction illustratedbelow for the Ras protein, in which the protein becomes attached to thefarnesyl group by displacement of pyrophosphate (P₂O₇ ⁴⁻, referred tohere as PP_(i)):

Thus, a key target in a strategy to retard cancer cell proliferation isthe enzyme FTase. By regulating FTase activity, Ras farnesylation, RhoBfarnesylation and geranylgeranylation, and the prenylation of otherproteins can be controlled. This can alter the intracellulardistribution of these proteins and in turn prevent cancer cells fromproliferating. Because normal cells also require FTase activity, theoptimal regulation of prenyltransferase activity must be determinedempirically.

Many substances are known to block FTase activity and preventfarnesylation of cellular proteins. These include inhibitors of theenzyme FTase, which generally operate by blocking the binding ofproteins to be prenylated, FPP, or both, to the FTase active site.Without the ability of the normal substrates (e.g. Ras and FPP) to bindto FTase, this enzyme can no longer transfer the farnesyl group from FPPto Ras. In general, inhibitors structurally mimic one or both of thenatural substrates of the enzyme, in this case Ras and/or FPP. Forconventional inhibitors, their binding to FTase is reversible andnoncovalent (i.e. the binding of the inhibitor to FTase does not involvethe formation of covalent bonds). Instead, hydrophobic forces, hydrogenbonding, electrostatic attraction, etc. are principally responsible forbinding of the inhibitor to the enzyme FTase. These binding forces allowthe inhibitor to block the site on FTase where the normal substratesneed to bind for farnesylation of Ras to occur.

It would therefore be desirable to develop a method of preventing,substantially irreversibly, FTase from farnesylating Ras and, moregenerally, preventing other prenylation enzymes from promoting the innercell membrane localization of oncoproteins. Interaction of FTase, forexample, with substances that covalently modify the active site of FTaseshould result in an enzyme with an essentially permanent reduction incatalytic ability. In principle, and in contrast to conventional enzymedeactivation, the covalent attachment can be irreversible or nearlyirreversible. The desirable characteristics of a prenylation enzymeinhibitor may include both a substrate-mimicking group as well as agroup having the ability to bond covalently to the enzyme at or near itsactive site.

SUMMARY OF THE INVENTION

The present invention is directed to methods for inhibiting aprenylation enzyme In one embodiment, the method comprises contacting aprenylation enzyme with a prenylation enzyme inhibitor of the followingstructural Formula I:

or a pharmaceutically acceptable salt, prodrug, or ester thereof,where A₁, A₂, and A₃ are independently selected from the groupconsisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryloxy,cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, haloalkyl,alkanoyl, aroyl, aminocarbonyl, aminoalkanoyl or optionally substitutedaminoalkanoyl, carbocycloalkyl or optionally substitutedcarbocycloalkyl, heterocyclo or optionally substituted heterocyclo,heteroaryl or optionally substituted heteroaryl, aryl, aralkyl,(heterocyclo)alkyl, (heteroaryl)alkyl, alkoxycarbonyl, alkylcarbonyloxy,alkoxyalkanoyl, and carboxyalkyl, and A₄ is selected from the groupconsisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryloxy,cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, haloalkyl,alkanoyl, aroyl, aminocarbonyl, aminoalkanoyl or optionally substitutedaminoalkanoyl, carbocycloalkyl or optionally substitutedcarbocycloalkyl, heterocyclo or optionally substituted heterocyclo,heteroaryl or optionally substituted heteroaryl, aryl, aralkyl,(heterocyclo)alkyl, (heteroaryl)alkyl, alkoxycarbonyl, alkoxyalkanoyl,carboxyalkyl, amino or substituted amino, amido or substituted amido,and alkanoylamido, with the proviso that A₁, A₂, A₃, and A₄ are not allhydrogen. In a preferred embodiment, A₁, A₂, A₃, and A₄ areindependently selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, aryl, aralkyl. In another preferred embodiment, atleast one of A₁, A₂, A₃, and A₄ is a branched alkenyl, which may bespecifically a farnesyl or geranylgeranyl group. In another preferredembodiment, the group A₄ is selected from the group consisting ofhydrogen, alkyl (e.g. methyl), haloalkyl (e.g. trichloromethyl,trifluoromethyl, perfluoroethyl), aryl (e.g. phenyl), and heteroaryl(e.g. 4-pyridyl).

In another preferred embodiment, the prenylation enzyme inhibitoraccording to the method has the following structural Formula II:

A-Y—(C=Z)-CX₁X₂X₃  (Formula II)

or a pharmaceutically acceptable salt, prodrug, or ester thereof,where Y is a heteroatom or heteroatomic group selected from the groupconsisting of O, NH, NA′, and S; where,when Y is O, A and A′ are independently selected from the groupconsisting of hydrogen, alkyl, alkoxyalkyl, alkylthio, alkenyl, alkynyl,cycloalkyl, haloalkyl, carbocycloalkyl or optionally substitutedcarbocycloalkyl, heterocyclo or optionally substituted heterocyclo,heteroaryl or optionally substituted heteroaryl, aryl, aralkyl,(heterocyclo)alkyl, (heteroaryl)alkyl, carboxyalkyl, amino orsubstituted amino, amido or substituted amido, and alkanoylamido;when Y is S, A and A′ are independently selected from the groupconsisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryloxy,cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, haloalkyl,carbocycloalkyl or optionally substituted carbocycloalkyl, heterocycloor optionally substituted heterocyclo, heteroaryl or optionallysubstituted heteroaryl, aryl, aralkyl, (heterocyclo)alkyl,(heteroaryl)alkyl, carboxyalkyl, amino or substituted amino, amido orsubstituted amido, and alkanoylamido; and,when Y is NH or NA′, A and A′ are independently selected from the groupconsisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryloxy,cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, haloalkyl,alkanoyl, aroyl, aminocarbonyl, aminoalkanoyl or optionally substitutedaminoalkanoyl, carbocycloalkyl or optionally substitutedcarbocycloalkyl, heterocyclo or optionally substituted heterocyclo,heteroaryl or optionally substituted heteroaryl, halo, aryl, aralkyl,(heterocyclo)alkyl, (heteroaryl)alkyl, alkoxycarbonyl, alkylcarbonyloxy,alkoxyalkanoyl, carboxyalkyl, amino or substituted amino, amido orsubstituted amido, and alkanoylamido;Z is oxygen or sulfur; and,X₁, X₂, and X₃ are independently selected from the group consisting ofhydrogen, oxygen, halogen, organosulfonyloxy, bromobenzenesulfonyloxy,methanesulfonyloxy, trifluoromethanesulfonyloxy, acyloxy, aryloxy,imidazolyl, —O—N═O, —NO₂, —OSO₃ ⁻, and —OPO₂(OH)⁻, with the proviso thatX₁, X₂, and X₃ are not all hydrogen. In a preferred embodiment, A, andoptionally A′, are independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl. In another preferredembodiment, at least one of A, and optionally A′, is a branched alkenyl,which may be specifically a farnesyl or geranylgeranyl group.Preferably, at least one of X₁, X₂, and X₃ is a nucleofugal groupcapable of bonding with the active site of a prenylation enzyme.

According to the possibilities for Formula II, there exists a carbonylvariation, where Z is oxygen, and also a thiocarbonyl variation, where Zis sulfur. In the case of the carbonyl variation, a preferred class ofprenylation inhibitors is obtained when X₁ is oxygen, such that theinhibitor will now have two carbonyl functions, and X₂ is methyl. Thesepreferred compounds may also be categorized as pyruvic acid derivatives.

In another embodiment, a method of screening compounds as potentialanti-tumor agents comprises contacting a prenylation enzyme with a testcompound according to Formula I or Formula II. The method furthercomprises measuring prenylation activity of the enzyme to identifycandidate anti-tumor agents. In a more specific embodiment, prior to thecontacting step, a natural substrate of the prenylation enzyme (e.g.farnesyl pyrophosphate and/or geranylgeranyl pyrophosphate) is added tothe test compound to compete therewith and indicate the specificity ofthe test compound for the prenylation enzyme.

In another embodiment, a method of inhibiting the growth of a cancercell comprises contacting the cancer cell with a prenylation enzymeinhibitor according to Formula I or Formula II, where the growth of thecancer cell is inhibited.

In another embodiment, a pharmaceutically acceptable formulationcomprises a compound according to Formula I or Formula II, and apharmaceutically acceptable carrier.

These and other embodiments are described below in the detaileddescription of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may hamper or prevent the proliferation of cancercells, possibly resulting in a decrease in tumor size and/ordisappearance of the cancer, to the benefit of cancer patients. It mayact by interference with cancer cell biochemistry, in which the enzymefarnesyl protein transferase, geranylgeranyl protein transferase, and/orsome other prenylation enzyme acts on the oncogenic Ras protein, RhoBprotein, or some other growth-related cellular protein. Alteration ofthe ratio of farnesylated RhoB to geranylgeranylated RhoB, for instance,through the action of farnesyl protein transferase inhibitors, isthought to have a profound effect on cancer cell proliferation. Thepresent invention may alter the farnesylation:geranylgeranylation ratiothrough the selective regulation of prenylation enzyme activity bycovalent modification of the active site of prenylation enzymes ratherthan reversible inhibition of these enzymes, as is the current practicewith farnesyl protein transferase reversible inhibitors.

The present invention is based on the effectiveness of prenylationenzyme inhibitors for ultimately reducing and/or terminating cancer cellproliferation through covalent or essentially irreversible modificationof the enzyme. The potential advantages of the present invention overreversible inhibitors of prenyltransferase are: (1) buildup of unusedsubstrate (e.g. FPP, Ras and RhoB proteins) cannot reverse the covalentbonding of this invention in the way reversible inhibitors can bedisplaced by unused substrates, making the present invention moreeffective; and (2) the effectiveness of periodic dosing with compoundsof the present invention, in contrast to the need for the constantpresence of reversible inhibitors, may (i) adjust the activity level ofthe enzyme, decreasing toxic side effects in the patient; and (ii)minimize the ability of the cancer cell to become resistant to thetherapy.

The present invention can reduce or eliminate the unrestrainedproliferation of cancer cells through the inhibition of enzymesaffecting biochemical reactions in these cells. The specificity of theinhibitor for the target prenylation enzyme can be optimized through twoparameters: (1) structural features, including structural similarity tothe normal substrates (e.g. FPP and/or Ras), that direct or otherwisefavor the binding of the inhibitor to the active site of enzyme, and (2)a reactivity that is appropriate for the chemical groups of theprenylation enzyme, particularly those that participate in the actualprocess catalyzed by the prenylation enzyme (e.g. those chemical groupsthat directly participate in the transfer of the farnesyl group of FPPto Ras). The structurally similar feature can be a hydrophobic componenthaving a high affinity for the active site of the enzyme. For example,the active site of FTase is hydrophobic, which favors binding to itsnatural substrate FPP, also containing a hydrophobic region (i.e. thefarnesyl group). Therefore, hydrophobic inhibitors that also contain analpha-oxo epoxide or a carbon atom bearing at least one nucleofugalatom, where the carbon atom is bonded to a carbonyl (C═O) group orthiocarbonyl (C═S) group, or masked form of an alpha-oxo epoxide,carbonyl, or thiocarbonyl group, such as a group that will convert to acarbonyl or thiocarbonyl group at physiological conditions, are in manycases sufficiently reactive with the prenylation enzyme to inhibit itsactivity.

The term “alkyl,” as used alone or in combination herein, refers to anunsubstituted or optionally substituted, straight, or branched chainsaturated hydrocarbon group containing from one to twenty-five carbonatoms, preferably from one to fifteen carbons, such as methyl, ethyl,n-propyl, n-butyl, pentyl, hexyl, heptyl, octyl, the various branchchain isomers thereof, such as isopropyl, isobutyl, sec-butyl,tert-butyl, isohexyl and the like. The alkyl group may be optionallysubstituted by one or more substituents, and generally no more thanthree, and most often just one substituent. Preferred optionalsubstituents include halo, alkoxy, amino, mono- and di-substitutedamino, aryl, carboxylic acid, heterocyclo, heteroaryl, cycloalkyl,hydroxy, trifluoromethoxy and the like.

The term “lower alkyl,” as used alone or in combination herein, refersto such alkyl groups containing from one to five carbon atoms.

The term “alkoxy,” as used alone or in combination herein, refers to analkyl group, as defined above, covalently bonded to the parent moleculethrough an —O— linkage, such as methoxy, ethoxy, propoxy, isopropoxy,butoxy, t-butoxy and the like.

The term “alkoxyalkyl,” as used alone or in combination herein, refersspecifically to an alkyl group substituted with an alkoxy group.

The term “aryloxy,” as used alone or in combination herein, refers to anaryl group, as defined below, covalently bonded to the parent moleculethrough an —O— linkage. An example of an aryloxy is phenoxy.

The term “cycloalkoxy,” as used alone or in combination herein, refersto a cycloalkyl group, as defined below, covalently bonded to the parentmolecule through an —O— linkage.

The term “alkylthio,” as used alone or in combination herein, refers toan alkyl group, as defined above, covalently bonded to the parentmolecule through an —S— linkage.

The term “alkenyl,” as used alone or in combination herein, refers to analkyl group, as defined above, containing one or more carbon-carbondouble bonds, preferably two or three double bonds. Examples of alkenylinclude ethenyl, propenyl, 1,3-butadienyl, and 1,3,5-hexatrienyl.

The term “alkynyl,” as used alone or in combination herein, refers to analkyl group, as defined above, containing one or more carbon-carbontriple bonds, preferably one or two such triple bonds.

The term “cycloalkyl,” as used alone or in combination herein, refers toan unsubstituted or optionally substituted, saturated cyclic hydrocarbongroup containing three to eight carbon atoms. The cycloalkyl group mayoptionally be substituted by one or more substituents, and generally nomore than three, and most often just one substituent. Preferred optionalsubstituents include alkyl, halo, amino, mono- and di-substituted amino,aryl, hydroxy and the like.

The term “haloalkyl,” as used alone or in combination herein, is aspecies of alkyl as defined herein, and particularly refers to an alkyl,preferably a lower alkyl, substituted with one or more halogen atoms,and preferably is a C₁ to C₄ alkyl substituted with one to three halogenatoms. One example of a haloalkyl is trifluoromethyl. Preferred examplesof haloalkyl groups include trichloromethyl, trifluoromethyl, andperfluoroethyl.

The term “alkanoyl,” as used alone or in combination herein, refers toan acyl radical derived from an alkanecarboxylic acid (alkyl-C(O)—),particularly a lower alkanecarboxylic acid, and includes such examplesas acetyl, propionyl, butyryl, valeryl, and 4-methylvaleryl.

The term “aroyl,” as used alone or in combination herein, means an acylradical derived from an aromatic carboxylic acid, such as optionallysubstituted benzoic or naphthoic acids and specifically includingbenzoyl and 1-naphthoyl.

The term “aminocarbonyl,” as used alone or in combination herein meansan amino-substituted carbonyl (carbamoyl or carboxamide) wherein theamino group is a primary amino (—NH₂). Substituted aminocarbonyl refersto secondary (mono-substituted amino) or tertiary amino (disubstitutedamino) group, as defined below, preferably having as a substituent(s) alower alkyl group.

The term “aminoalkanoyl,” as used alone or in combination herein, meansan amino-substituted alkanoyl wherein the amino group is a primary aminogroup (-alkyl-C(O)—NH₂). The term “substituted aminoalkanoyl” refers torelated secondary (mono-substituted amino) or tertiary amino(di-substituted amino) group, as defined below.

The term “carbocycloalkyl,” as used alone or in combination herein,refers to an unsubstituted or optionally substituted, stable, saturatedor partially unsaturated monocyclic, bridged monocyclic, bicyclic, orspiro ring carbocycle of 3 to 15 carbon atoms such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclohexyl,bicyclooctyl, bicyclononyl, spirononyl and spirodecyl. Cycloalkyls arethus one specific subset of carbocycloalkyls. The term “optionallysubstituted” as it refers to “carbocycloalkyl” herein indicates that thecarbocycloalkyl group may be substituted at one or more substitutablering positions by one or more groups independently selected from alkyl(preferably lower alkyl), alkoxy (preferably lower alkoxy), nitro,monoalkylamino (preferably a lower alkylamino), dialkylamino (preferablya di[lower]alkylamino), cyano, halo, haloalkyl (preferablytrifluoromethyl), alkanoyl, aminocarbonyl, monoalkylaminocarbonyl,dialkylaminocarbonyl, alkylamido, (preferably a lower alkylamido),alkoxyalkyl (preferably a lower alkoxy[lower]alkyl), alkoxycarbonyl(preferably a lower alkoxycarbonyl), alkylcarbonyloxy (preferably alower alkylcarbonyloxy) and aryl (preferably phenyl), said aryl beingoptionally substituted by halo, lower alkyl and/or lower alkoxy groups.Generally, there is no more than one optional substituent.

The term “heterocyclo,” as used alone or in combination herein, refersto an unsubstituted or optionally substituted, stable, saturated, orpartially unsaturated, monocyclic, bridged monocyclic, bicyclic, orspiro ring system containing carbon atoms and other atoms selected fromnitrogen, sulfur and/or oxygen. Preferably, a heterocyclo group is a 5or 6-membered monocyclic ring or an 8-11 membered bicyclic ring thatconsists of carbon atoms and contains one, two, or three heteroatomsselected from nitrogen, oxygen and/or sulfur. Heterocyclo includesbent-fused monocyclic cycloalkyl groups having at least one suchheteroatom. The term “optionally substituted,” as it refers to“heterocyclo” herein, indicates that the heterocyclo group may besubstituted at one or more substitutable ring positions by one or moregroups independently selected from alkyl (preferably lower alkyl andincluding haloalkyl (preferably trifluoromethyl)), alkoxy (preferablylower alkoxy), nitro, monoalkylamino (preferably a lower alkylamino),dialkylamino (preferably a di[lower]alkylamino), cyano, halo, alkanoyl,aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, alkylamido(preferably lower alkylamido), alkoxyalkyl (preferably a loweralkoxy[lower]alkyl), alkoxycarbonyl (preferably a lower alkoxycarbonyl),alkylcarbonyloxy (preferably a lower alkylcarbonyloxy) and aryl(preferably phenyl), said aryl being optionally substituted by halo,lower alkyl and lower alkoxy groups. Generally, there is no more thanone optional substituent. The heterocyclo group may be, and generallyis, attached to the parent structure through a carbon atom, oralternatively may be attached through any heteroatom of the heterocyclogroup that results in a stable structure.

The term “heteroaryl,” as used alone or in combination herein, refers toan unsubstituted or optionally substituted, stable, aromatic monocyclicor bicyclic ring system containing carbon atoms and other atoms selectedfrom nitrogen, sulfur and/or oxygen. Preferably, a heteroaryl group is a5- or 6-membered monocyclic ring (optionally benzofused) or an 8-11membered bicyclic ring that consists of carbon atoms and contains one,two, or three heteroatoms selected from nitrogen, oxygen and/or sulfur.The term “optionally substituted” as it refers to “heteroaryl” hereinindicates that the heteroaryl group may be substituted at one or moresubstitutable ring positions by one or more groups independentlyselected from alkyl (preferably lower alkyl and including haloalkyl(preferably trifluoromethyl)), alkoxy (preferably lower alkoxy), nitro,monoalkylamino (preferably a lower alkylamino), dialkylamino,(preferably a di[lower]alkylamino), cyano, halo, alkanoyl,aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, alkylamido(preferably lower alkylamido), alkoxyalkyl (preferably a loweralkoxy[lower]alkyl), alkoxycarbonyl (preferably a lower alkoxycarbonyl),alkylcarbonyloxy (preferably a lower alkylcarbonyloxy), and aryl(preferably phenyl), said aryl being optionally substituted by halo,lower alkyl, and lower alkoxy groups. Generally, there is no more thanone optional substituent. The heteroaryl group may be, and generally isattached to the parent structure through a carbon atom or alternativelymay be attached through any heteroatom of the heteroaryl group thatresults in a stable structure. In the foregoing structures it is alsocontemplated that a nitrogen could be replaced with an N-oxide. Bothheterocyclo and heteroaryl also are intended to embrace benzo fusedstructures such as 1,2-methylenedioxybenzene and 1,4-benzodioxan.Preferred examples of heteroaryl groups include pyridyl (e.g. 2-, 3-, or4-pyridyl).

The terms “halo” and “halogen,” as used alone or in combination herein,represent fluorine, chlorine, bromine or iodine, preferably fluorine orchlorine for enzyme affinity, and preferably chlorine, bromine, oriodine when a nucleofuge.

The term “aryl,” as used alone or in combination herein, refers to anunsubstituted or optionally substituted monocyclic or bicyclic aromatichydrocarbon ring system having 6 to 12 ring carbon atoms. Preferred areoptionally substituted phenyl, 1-naphthyl, or 2-naphthyl groups. Thearyl group may optionally be substituted at one or more substitutablering positions (generally at no more than three positions and most oftenat one or two positions) by one or more groups independently selectedfrom alkyl (including haloalkyl (preferably trifluoromethyl anddifluoromethyl)), alkenyl, alkynyl, alkoxy, aryloxy, nitro, hydroxy,amino, mono- and di-substituted amino, cyano, halo, alkanoyl,aminocarbonyl, carboxylic acid, carboxylic acid esters, carboxylic acidamide, an optionally substituted phenyl (optionally substituted by halo,lower alkyl and lower alkoxy groups), heterocyclo, or heteroaryl.Preferably, the aryl group is phenyl optionally substituted with up tofour and more usually with one or two groups, preferably selected fromlower alkyl, lower alkoxy, as well as cyano, trifluoromethyl, and halo.

The terms “aralkyl” and “(aryl)alkyl,” as used alone or in combinationherein, are species of alkyl as defined herein, and particularly referto an alkyl group as defined above in which one hydrogen atom isreplaced by an aryl group as defined above, and include benzyl, and2-phenylethyl.

The terms “(heterocyclo)alkyl” and “(heteroaryl)alkyl,” as used alone orin combination can be considered a species of alkyl as defined herein,and particularly refer to an to an alkyl group as defined above in whichone hydrogen atom is replaced by a heterocyclo group as defined above,or by a heteroaryl group as defined above.

The terms “alkoxycarbonyl,” as used alone or in combination herein, meana radical of the formula —C(O)-alkoxy, in which alkoxy is as definedabove.

The term “alkylcarbonyloxy,” as used alone or in combination herein,means a radical of the formula —O—C(O)-alkyl, in which alkyl is asdefined above.

The term “alkoxyalkanoyl,” as used alone or in combination herein, meansa radical of the formula -alkyl-C(O)—O-alkyl.

The term “carboxyalkyl,” as used alone or in combination herein, means aradical of the formula -alkyl-C(O)—OH.

The term “substituted amino,” as used alone or in combination herein,embraces both mono and di-substituted amino. These terms, alone, or incombination, mean a radical of the formula —NR′R″, where, in the case ofmono-substitution, one of R′ and R″ is a hydrogen and the other isselected from alkyl, cycloalkyl, aryl, heterocyclo, (aryl)alkyl,(heterocyclo)alkyl, heteroaryl and hetero(aryl)alkyl; in the case ofdi-substitution, R′ and R″ are independently selected from alkyl,cycloalkyl, aryl, heterocyclo, and heteroaryl, or R′ and R″ togetherwith the nitrogen atom to which they are both attached form a three toeight-membered heterocyclo or heteroaryl radical.

The term “amido,” as used alone or in combination herein, refers to thegroup (—NH—) and the term “substituted amido” embraces a radical of theformula (—NR′—) where R′ has the meaning above in connection withsubstituted amino.

The terms “alkanoylamido,” “aroylamido,” “heterocyclocarbonylamido” and“heteroaroylamido,” as used alone or in combination herein, mean groupsof the formula R—C(O)—NH— where R is an alkyl, aryl, heteroaryl orheterocyclo group. The terms “heteroaroyl” and “heterocyclocarbonyl,”when used alone or in combination, mean groups of the formula R—C(O)—where R is a heteroaryl or heterocyclo group.

Unless otherwise defined, the term “optionally substituted” as usedherein, refers to the substitution of a ring system at one or morepositions with one or more groups selected from: C₁-C₅ alkyl, C₁-C₅alkoxy, an optionally substituted phenyl, cyano, halo, trifluoromethyl,C₁-C₅ alkoxycarbonyl, C₁-C₅ alkyl carbonyloxy, mono- andbis-(C₁-C₅alkyl)-carboxamide, C₁-C₅ alkylamido, nitro, and mono- andbis-(C₁-C₅ alkyl)amino.

The terms “hydrophobic group” and “hydrophobic component” as usedherein, refer to any of the groups hydrogen, alkyl, alkoxy, alkoxyalkyl,aryloxy, cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl,haloalkyl, alkanoyl, aroyl, aminocarbonyl, aminoalkanoyl or optionallysubstituted aminoalkanoyl, carbocycloalkyl or optionally substitutedcarbocycloalkyl, heterocyclo or optionally substituted heterocyclo,heteroaryl or optionally substituted heteroaryl, halo, aryl, aralkyl,(heterocyclo)alkyl, (heteroaryl)alkyl, alkoxycarbonyl, alkylcarbonyloxy,alkoxyalkanoyl, carboxyalkyl, amino or substituted amino, amido orsubstituted amido, and alkanoylamido as defined above having at leastsome affinity for a hydrocarbon.

The terms “nucleofugal atom” and “nucleofugal group” as used hereinrefer to reactive leaving groups that, after reaction, can depart with alone pair of electrons. Nucleofugal atoms or groups include halogenatoms (e.g. F, Cl, Br, and I) organosulfonyloxy groups (e.g.p-toluenesulfonyloxy, p-bromobenzenesulfonyloxy, methanesulfonyloxy,trifluoromethanesulfonyloxy, etc.), acyloxy groups (e.g. CH₃CO₂—,CCl₃CO₂—), aryloxy groups (e.g. phenyl-O—), imidazolyl groups, —O—N═O,—NO₂, —OSO₃ ⁻, and —OPO₂(OH)⁻.

It is recognized that there may be some overlap in some of thedefinitions of the various groups. Specific groups are mentioned,however, and may be particularly identified in the claims, in order toemphasize their positive inclusion in the described subject matter, asnot only an optional substituent. As used herein, when a particulargroup, generally understood to have a single point of attachment to acore structure, such as an alkyl group, is identified in connection witha structure that must have two points of attachment in the structuralcore, it is understood that the named group (e.g., alkyl) refers to theparent group with a hydrogen or a site of unsaturation removed to createthe second point of attachment so as to provide the required structure.

Compounds according to one embodiment of the present invention have thefollowing generalized structural Formula I:

or a pharmaceutically acceptable salt, prodrug, or ester thereofwhere A₁, A₂, and A₃, are independently selected from the groupconsisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryloxy,cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, haloalkyl,alkanoyl, aroyl, aminocarbonyl, aminoalkanoyl or optionally substitutedaminoalkanoyl, carbocycloalkyl or optionally substitutedcarbocycloalkyl, heterocyclo or optionally substituted heterocyclo,heteroaryl or optionally substituted heteroaryl, aryl, aralkyl,(heterocyclo)alkyl, (heteroaryl)alkyl, alkoxycarbonyl, alkylcarbonyloxy,alkoxyalkanoyl, and carboxyalkyl, and A₄ is selected from the groupconsisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryloxy,cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, haloalkyl,alkanoyl, aroyl, aminocarbonyl, aminoalkanoyl or optionally substitutedaminoalkanoyl, carbocycloalkyl or optionally substitutedcarbocycloalkyl, heterocyclo or optionally substituted heterocyclo,heteroaryl or optionally substituted heteroaryl, aryl, aralkyl,(heterocyclo)alkyl, (heteroaryl)alkyl, alkoxycarbonyl, alkoxyalkanoyl,carboxyalkyl, amino or substituted amino, amido or substituted amido,and A₁, A₂, A₃, and A₄ are not all hydrogen, since this would result ina compound lacking any specificity to the enzyme compared to the desiredcondition where at least one of these pendant groups has at least somespecificity.

Preferably, at least one of A₁, A₂, A₃, and A₄ is a hydrophobiccomponent designed to impart specificity of the substance for binding toand/or inactivation of FTase or GGTase. In this respect, in a preferredembodiment, A₁, A₂, A₃, and A₄ are independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl. Forexample, at least one of A₁, A₂, A₃, and A₄ is a branched alkenyl, whichmight be specifically a farnesyl or geranylgeranyl group. In this case,the prenylation enzyme inhibitor has a pendant group that matches thatof the substrates for which FTase and GGTase, respectively, haveaffinity. In another preferred embodiment, the group A₄ is selected fromthe group consisting of hydrogen, alkyl (e.g. methyl), haloalkyl (e.g.trichloromethyl, trifluoromethyl, perfluoroethyl), aryl (e.g. phenyl),and heteroaryl (e.g. 4-pyridyl). These particular groups for A₄ resultin an inhibitor having improved ability to react with the active site ofa prenylation enzyme. More specifically, and without being bound to anyparticular theory, the presence of halogen atoms in the group A₄enhances the reactivity of the inhibitor with epsilon amino groups foundin the prenylation enzyme active sites, and particularly the lysineepsilon amino group found in the active site of FTase. Additionally, thepresence of phenyl and pyridyl groups enhance the stability of a Schiffbase or imine that is believed to result from reaction of the inhibitorwith the enzyme. From the above explanation, a particularly preferredvariant of the prenylation enzyme inhibitor of the present inventionaccording to Formula I is one in which at least one of A₁, A₂, and A₃ isa hydrophobic group (e.g. a farnesyl or a geranylgeranyl group) and A₄is selected from the group consisting of alkyl, haloalkyl, aryl, andheteroaryl.

Possible methods of synthesizing prenylation inhibitor compounds of thepresent invention are provided below for compounds according to FormulaI and Formula II as described above. In the case of alpha-oxo epoxideswithin the scope of Formula I, the treatment of thealpha,beta-unsaturated carbonyl compound with alkaline hydrogenperoxide, is depicted below:

Another synthesis possibility for compounds of Formula I is theSharpless epoxidation of the allylic alcohol, followed by oxidation ofthe alcohol. This method results in the stereospecific formation ofchiral alpha-oxo epoxides, and is depicted below:

Another class of compounds contemplated within the scope of theinvention has the following generalized structural Formula II:

A-Y—(C=Z)-CX₁X₂X₃  (Formula II)

or a pharmaceutically acceptable salt, prodrug, or ester thereofwhere Y is a heteroatom or heteroatomic group selected from the groupconsisting of O, NH, NA′, and S; where,when Y is O, A and A′ are independently selected from the groupconsisting of hydrogen, alkyl, alkoxyalkyl, alkylthio, alkenyl, alkynyl,cycloalkyl, haloalkyl, carbocycloalkyl or optionally substitutedcarbocycloalkyl, heterocyclo or optionally substituted heterocyclo,heteroaryl or optionally substituted heteroaryl, aryl, aralkyl,(heterocyclo)alkyl, (heteroaryl)alkyl, carboxyalkyl, amino orsubstituted amino, amido or substituted amido, and alkanoylamido;when Y is S, A and A′ are independently selected from the groupconsisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryloxy,cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, haloalkyl,carbocycloalkyl or optionally substituted carbocycloalkyl, heterocycloor optionally substituted heterocyclo, heteroaryl or optionallysubstituted heteroaryl, aryl, aralkyl, (heterocyclo)alkyl,(heteroaryl)alkyl, carboxyalkyl, amino or substituted amino, amido orsubstituted amido, and alkanoylamido; and,when Y is NH or NA′, A and A′ are independently selected from the groupconsisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryloxy,cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, haloalkyl,alkanoyl, aroyl, aminocarbonyl, aminoalkanoyl or optionally substitutedaminoalkanoyl, carbocycloalkyl or optionally substitutedcarbocycloalkyl, heterocyclo or optionally substituted heterocyclo,heteroaryl or optionally substituted heteroaryl, halo, aryl, aralkyl,(heterocyclo)alkyl, (heteroaryl)alkyl, alkoxycarbonyl, alkylcarbonyloxy,alkoxyalkanoyl, carboxyalkyl, amino or substituted amino, amido orsubstituted amido, and alkanoylamido;Z is O or S, resulting in a carbonyl or thiocarbonyl group; and,X₁, X₂, and X₃ are independently selected from the group consisting ofhydrogen, halogen, organosulfonyloxy, bromobenzenesulfonyloxy,methanesulfonyloxy, trifluoromethanesulfonyloxy, acyloxy, aryloxy,imidazolyl, —O—N═O, —NO₂, —OSO₃ ⁻, and —OPO₂(OH)⁻. The groups X₁, X₂,and X₃ are not all hydrogen, as this particular case would result in thecompound having an attached methyl group which is relatively unreactivecompared to the desired condition where at least one of these pendantgroups is a nucleofugal group. In a preferred embodiment, A, andoptionally A′, are independently selected from the group consisting ofhydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl. In another preferredembodiment, at least one of A, and optionally A′ is a branched alkenyl,which may be specifically a farnesyl or geranylgeranyl group.

According to the possibilities for Formula II, there exists a carbonylvariation, where Z is oxygen, and also a thiocarbonyl variation, where Zis sulfur. Preferred enzyme inhibitor compounds within the generalizedstructural Formula II can be characterized as alpha-haloesters oralpha-halothioesters where Y is an oxygen atom, X₁ and X₂ are hydrogen,and X₃ is a halogen. In the case of the carbonyl variation of FormulaII, a preferred class of prenylation inhibitors is obtained when X₁ isoxygen, such that the inhibitor will now have two carbonyl functions,and X₂ is methyl. These preferred compounds may also be categorized aspyruvic acid derivatives.

As mentioned, the portion of the compound represented by any of A aidsin the selective, noncovalent binding or affinity of the inventivecompounds for prenyltransferases. Also, the carbonyl or thiocarbonylgroup of compounds represented by Formula II, combined with an adjacentnucleofuge-bearing carbon atom, can subsequently bond to theprenyltransferase active site, thereby regulating the level of activityof the prenyltransferase by hampering access of substrates to the activesite residues and/or other mechanisms such as by inducing conformationalchanges in the prenyltransferase that affect its catalytic ability. Theregulation of catalytic activity of the prenyltransferase can also beachieved by total inactivation of a portion of the prenyltransferasemolecules while leaving some molecules completely unmodified.

It is possible to prepare compounds within the scope of Formula TIaccording to the pathway provided below, where, for example, A is afarnesyl substitutent, Y and Z are oxygen, X₁ and X₂ are hydrogen, andX₃ is bromine:

The pathway outlined below depicts the synthesis of another compound ofFormula II, having a hydrophobic substituent comprising a phenyl groupand a polyether functionality:

Other embodiments of the invention include a pharmaceutically acceptablesalt, prodrug, or ester of the compounds of Formula I or Formula II. Byway of example, for compounds having structural Formula TI, the carbonylgroups can be masked in various forms including a hydrate [C(OH)₂], ahemiacetal or hemiketal [C(OH)(OR′)], an acetal or a ketal[C(OR′)(OR″)], an acylal or related compound [C(OC(═O)R′)OC(═O)R″)], abisulfite addition compound [C(OH)(SO₃ ⁻)], an enol (C═COH), an enolether (C═COR′), an enol ester [C═COC═(═O)R′], and so forth, wherein R′and R″ are independently selected from the group consisting of hydrogen,alkyl, alkoxy, alkoxyalkyl, aryloxy, cycloalkoxy, alkylthio, alkenyl,alkynyl, cycloalkyl, haloalkyl, alkanoyl, aroyl, aminocarbonyl,aminoalkanoyl or optionally substituted aminoalkanoyl, carbocycloalkylor optionally substituted carbocycloalkyl, heterocyclo or optionallysubstituted heterocyclo, heteroaryl or optionally substitutedheteroaryl, halo, aryl, aralkyl, (heterocyclo)alkyl, (heteroaryl)alkyl,alkoxycarbonyl, alkylcarbonyloxy, alkoxyalkanoyl, carboxyalkyl, amino orsubstituted amino, amido or substituted amido, and alkanoylamido.Likewise, prenylation enzyme inhibitors according to the thiocarbonylvariation of Formula II may also include masked thiocarbonyl groups, inwhich the oxygen atoms in the masked groups according to the abovedescription are replaced by sulfur atoms. Of course, it is possiblethat, where the masked groups according to the above description containmore than one oxygen atom, a combination of oxygen and sulfur atoms mayactually be included in the masked group. Such masked carbonyl orthiocarbonyl groups may produce the carbonyl or thiocarbonyl groups ofthe prenylation enzyme inhibitors of the present invention underphysiological conditions.

Some cancer cells in which farnesylation of Ras is blocked alternativelyemploy the related prenylation reaction geranylgeranylation to attach ahydrophobic group to Ras, accomplishing membrane localization andcontinued cancerous behavior of the cell. The enzyme that attaches thegeranylgeranyl group to Ras protein to facilitate localization at theinner surface of the cancer cell membrane is geranylgeranyl proteintransferase, also known as protein geranylgeranyl transferase (referredto here as GGTase). The geranylgeranyl group becomes attached to Ras byreaction with geranylgeranyl diphosphate, also known as geranylgeranylpyrophosphate (referred to here as GGPP). Stated otherwise, GGTasecatalyzes the following reaction, in which Ras becomes attached togeranylgeranyl group by displacement of pyrophosphate (P₂O₇ ⁴⁻, referredto here as PP_(i)):

The newly formed geranylgeranyl-Ras localizes at the inner surface ofthe cancer cell membrane and causes the cancer cell to divide withoutrestraint. Thus, a key target in a strategy to retard cancer cellproliferation is the enzyme GGTase. By reducing or destroying GGTaseactivity, either in combination with regulation of Ras farnesylation orindependently, Ras geranylgeranylation may be also regulated, which inturn should further hinder the ability of the cancer cell to divide andproliferate through localization of Ras at its inner membrane surface.As noted previously, regulation of RhoB geranlygeranylation by use ofinhibitors of the present invention is also contemplated as a means forretarding and/or terminating cancer cell growth.

To more precisely align the structure of the inhibitors of the presentinvention with the active sites of FTase, GGTase, or other enzymes,variation of the distance between the covalent-bonding group and thefarnesyl-mimicking or geranylgeranyl-mimicking group is achieved throughaltering the length of a “spacer” between such groups. For example,inhibitors representative of the carbonyl variation of structuralFormula TI may be more precisely tailored to inhibit either FTase orGGTase activity by altering the spacer length as represented below:

where the value of n, representing the number of carbon atoms betweenthe respective farnesyl and geranylgeranyl groups, will generally rangefrom 0 to about 10, although instances where longer chains are requiredfor alignment with other types of enzymes are readily recognizable tothe ordinary skilled artisan having regard for this disclosure.

Inhibitors of the present invention may also incorporate an aromaticgroup for enhanced binding to the hydrophobic binding site of FTase orGGTase. Such compounds are exemplified by the following specific enzymeinhibitors according to the carbonyl variation of structural Formula II,although numerous other possible embodiments of this type of compoundare of course possible and readily apparent to one of ordinary skill inthe art, having regard for this disclosure:

Three additional possibilities are represented below, where the last twohave an A group that is selective for the FTase active site over theGGTase and squalene synthase active sites, thus conferring addedspecificity for the desired target enzyme.

The inhibitors of the present invention are applicable in particular tothe reduction in prenylation activity of the enzymes farnesyl proteintransferase and geranylgeranyl protein transferase. Without wishing tobe bound by any particular theory or reaction mechanism, a hypotheticalpathway illustrating FTase inhibition using the alpha-oxo epoxideprenylation enzyme inhibitors, according to Formula I of the presentinvention, is shown below:

As shown, the FTase active site contains two amino acid residues thatare involved in the reaction with the inhibitor, namely His-248 andLys-294. The rest of the enzyme is conveniently represented as E. Thesetwo residues are in close proximity as a consequence of their normalrole in binding the natural substrate FPP at its terminal phosphate. Asshown above, nucleophilic attack by the H is imidazole group results inepoxide ring opening, thus making the epoxide oxygen formally anucleofuge. In addition to alkylation of the imidazole ring, the nearbyepsilon-amino group of the Lys forms a Schiff base or imine with thealdehyde group of the alpha-oxo epoxide. The result of the abovereactions is the crosslinking of two active site residues. The resultingcrosslinked structure irreversibly precludes the FTase active siteresidues from catalyzing prenylation reactions, thus impairing theenzyme's overall functioning.

Analogous reaction mechanisms can be postulated for inhibitor compoundsof the present invention according to both the carbonyl and thiocarbonylvariations of Formula II. In these cases, the nucleofugal atom or groupadjacent to a carbonyl group can react with the aforementioned activesite residues to crosslink them in an analogous manner to the mechanismshown above, with an accompanying release of the nucleofugal group.

Preferably, the inhibitor is administered under proper conditions and ina concentration such that its presence in the prenylation system reducesprenylation activity by at least about 50%, more preferably at leastabout 75%, and even more preferably by at least about 90%.Pharmaceutical formulations can be prepared by combining appropriateamounts of the inhibitor in a pharmaceutically acceptable carrier,diluent, or excipient. In such formulations, the inhibitor is typicallypresent in an amount from about 0.1-20% by weight, and more commonlyfrom about 1-10%.

Combinations of this invention with other anticancer agents to producesynergistic effects of benefit to the patient are also possible. Thismight be based on two strategies. One is to interfere with differentbiochemical processes to increase tumor cell killing. Another is tohamper development of drug resistance, which is less likely to occursimultaneously in tumor cells exposed to anticancer agents based oninterference with different biochemical pathways in the tumor cells.

In summary an improved method of interference with protein prenylationin tumor cells has been described that may prevent or hamper theproliferation of tumor cells, possibly resulting in a decrease in tumorsize and/or disappearance of the cancer, to the benefit of cancerpatients.

EXAMPLES 1-2

Two alpho-oxo epoxides according to Formula I of the present inventionwere synthesized according to procedures set forth in H. Yao and D. E.Richardson, J. Am. Chem. Soc. 2000, 122, 3220-3221 and also in C. A.Bunton and G. J. Minkoff, J. Chem. Soc. 1949, 665-670. The compoundswere then tested for their inhibition of the growth of various cancercells. These compounds had, as their A₂ substituent, farnesyl andgeranylgeranyl groups, respectively. These inhibitors had structuralformulas as shown below.

The compounds were tested for growth inhibition in a variety of humancancer cell lines, according to procedures described by K. J.Okolotowicz, W. L. Lee, R. F. Hartman, S. R. Lefler, and S. D. Rose,Inactivation of Protein Farnesyltransferase by Active-Site-TargetedDicarbonyl Compounds, Arch. Pharm. Pharm. Med. Chem. 2001, 334, 194-202.Results of biological testing of these compounds are summarized in Table1, where the GI₅₀ value represents the concentration of inhibitorcompound required to effect a 50% reduction in cell growth (i.e. 50%inhibition).

TABLE 1 Inhibition of Cancer Cell Growth by Alpha-oxo EpoxidesGI₅₀/molar GI₅₀/niolar Human cancer cell line Compound RG-22 CompoundRG-23 Breast (MCF-7) 14 × 10⁻⁶ 8.9 × 10⁻⁶  Prostate (DU-145) 27 × 10⁻⁶28 × 10⁻⁶ Central nervous syst.(SF268) 30 × 10⁻⁶ 42 × 10⁻⁶ Pancreatic(BXPC-3) 67 × 10⁻⁶ 51 × 10⁻⁶ Colon (KM20L2) 95 × 10⁻⁶ 61 × 10⁻⁶ Lung(NCI-H460) 117 × 10⁻⁶  63 × 10⁻⁶

Results show that both compounds are extremely active for inhibitingcell growth in various human cancer cell lines in culture. In fact, oneof the GI₅₀ values was below 10 micromoles/liter. Additionally, thecompounds were active against mouse P388 leukemia cells in culture. Inthese cases, compound RG-22 exhibited an ED₅₀ of 26×10⁻⁶ M, and RG-23exhibited an ED₅₀ of only 11×10⁻⁶ M. Based on these results, theanticancer activity of these compounds is confirmed.

EXAMPLES 3-4

Two alpha-halo carbonyl compounds were also synthesized as describedpreviously herein and tested for their inhibition of the growth ofvarious cancer cells. These compounds had substituent farnesyl groups aswell as substituent nucleofugal halogen atoms. The inhibitors hadstructural formulas as shown below:

The compounds were tested for growth inhibition in a variety of humancancer cell lines, as described in Examples 1-2. Results of the in vitrocell culture testing of these compounds are summarized in Table 2, whichprovides the associated GI₅₀ values.

TABLE 2 Cell Culture Results with Alpha-halo Carbonyl Compounds Example3 Example 4 Human cancer cell line GI₅₀/molar GI₅₀/molar Colon (HT-29)10⁻⁴ to 10⁻⁵ 10⁻⁴ to 10⁻⁵ Colorectal (Colo-205) 10⁻⁴ to 10⁻⁵ 10⁻⁴ to10⁻⁵ NSC Lung (H-460) 1.1 × 10⁻⁵ 1.4 × 10⁻⁵ Prostate (PC-3) 7.9 × 10⁻⁶1.6 × 10⁻⁵ Acute myeloid leukemia (HL-60) Above 10⁻⁴  10⁻⁹ to 10⁻¹⁰Fibrosarcoma (HT-1080) 10⁻⁵ to 10⁻⁶ 10⁻⁴ to 10⁻⁵ Urinary bladder (T-24)10⁻⁴ to 10⁻⁵ 10⁻⁴ to 10⁻⁵ Colon (Caco-2) Above 10⁻⁴ 10⁻⁴ to 10⁻⁵

Again, results show that the compounds provide effective inhibition ofcancer cell growth, even at low concentrations.

1-7. (canceled)
 8. A method of screening compounds as potentialanti-tumor agents, the method comprising contacting a prenylation enzymewith a test compound of Formula II:A-Y—(C=Z)-CX₁X₂X₃  (Formula II) or a pharmaceutically acceptable salt,prodrug, or ester thereof, wherein: Z is O or S; Y is a heteroatom orheteroatomic group selected from the group consisting of O, NH, NA′, andS; wherein, when Y is O, A is selected from the group consisting ofhydrogen, alkyl, alkoxyalkyl, alkylthio, alkenyl, alkynyl, cycloalkyl,haloalkyl, carbocycloalkyl- or optionally substituted carbocycloalkyl,heterocyclo or optionally substituted heterocyclo, heteroaryl oroptionally substituted heteroaryl, aryl, aralkyl, (heterocyclo)alkyl,(heteroaryl)alkyl, carboxyalkyl, amino or substituted amino, amido orsubstituted amido, and alkanoylamido; when Y is S, A is selected fromthe group consisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryloxy,cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, haloalkyl,carbocycloalkyl or optionally substituted carbocycloalkyl, heterocycloor optionally substituted heterocyclo, heteroaryl or optionallysubstituted heteroaryl, aryl, aralkyl, (heterocyclo)alkyl,(heteroaryl)alkyl, carboxyalkyl, amino or substituted amino, amido orsubstituted amido, and alkanoylamido; and, when Y is NH or NA′, A and A′are independently selected from the group consisting of hydrogen, alkyl,alkoxy, alkoxyalkyl, aryloxy, cycloalkoxy, alkylthio, alkenyl, alkynyl,cycloalkyl, haloalkyl, alkanoyl, aroyl, aminocarbonyl, aminoalkanoyl oroptionally substituted aminoalkanoyl, carbocycloalkyl or optionallysubstituted carbocycloalkyl, heterocyclo or optionally substitutedheterocyclo, heteroaryl or optionally substituted heteroaryl, halo,aryl, aralkyl, (heterocyclo)alkyl, (heteroaryl)alkyl, alkoxycarbonyl,alkylcarbonyloxy, alkoxyalkanoyl, carboxyalkyl, amino or substitutedamino, amido or substituted amido, and alkanoylamido, and X₁, X₂, and X₃are independently selected from the group consisting of hydrogen,halogen, organosulfonyloxy, bromobenzenesulfonyloxy, methanesulfonyloxy,trifluoromethanesulfonyloxy, acyloxy, aryloxy, imidazolyl, —O—N═O, —NO₂,—OSO₃ ⁻, and —OPO₂(OH)⁻, with the proviso that X₁, X₂, and X₃ are notall hydrogen, and measuring prenylation activity of the enzyme, whereina reduction in prenylation activity renders the test compound acandidate anti-tumor agent.
 9. The method of claim 8, wherein Z is O.10. The method of claim 8, wherein Z is S.
 11. The method of claim 8,wherein the reduction in prenylation activity is at least about 50% torender the test compound a candidate anti-tumor agent.
 12. The method ofclaim 11, wherein the reduction in prenylation activity is at leastabout 75% to render the test compound a candidate anti-tumor agent. 13.The method of claim 12, wherein the reduction in prenylation activity isat least about 90% to render the test compound a candidate anti-tumoragent.
 14. The method of claim 8, wherein the prenylation enzyme isfarnesyl protein transferase or geranylgeranyl protein transferase. 15.The method of claim 8, wherein a natural substrate of the prenylationenzyme is added to the test compound to compete therewith and indicatethe specificity of the test compound for the prenylation enzyme.
 16. Themethod of claim 15, wherein the natural substrate of the prenylationenzyme is farnesyl pyrophosphate or geranylgeranyl pyrophosphate. 17-25.(canceled)
 26. A pharmaceutical formulation comprising a compound ofFormula II:A-Y—(C=Z)-CX₁X₂X₃  (Formula II) or a pharmaceutically acceptable salt,prodrug, or ester thereof, and a pharmaceutically acceptable carrier,diluent, or excipient, wherein: Z is O or S; Y is a heteroatom orheteroatomic group selected from the group consisting of O, NA′, and S;wherein, when Y is O, A is selected from the group consisting ofhydrogen, alkyl, alkoxyalkyl, alkylthio, alkenyl, alkynyl, cycloalkyl,haloalkyl, carbocycloalkyl- or optionally substituted carbocycloalkyl,heterocyclo or optionally substituted heterocyclo, heteroaryl oroptionally substituted heteroaryl, aryl, aralkyl, (heterocyclo)alkyl,(heteroaryl)alkyl, carboxyalkyl, amino or substituted amino, amido orsubstituted amido, and alkanoylamido; when Y is S, A is selected fromthe group consisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryloxy,cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, haloalkyl,carbocycloalkyl or optionally substituted carbocycloalkyl, heterocycloor optionally substituted heterocyclo, heteroaryl or optionallysubstituted heteroaryl, aryl, aralkyl, (heterocyclo)alkyl,(heteroaryl)alkyl, carboxyalkyl, amino or substituted amino, amido orsubstituted amido, and alkanoylamido; and, when Y is NA′, A is selectedfrom the group consisting of hydrogen, alkyl, alkoxy, alkoxyalkyl,aryloxy, cycloalkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl,haloalkyl, alkanoyl, aroyl, aminocarbonyl, aminoalkanoyl or optionallysubstituted aminoalkanoyl, carbocycloalkyl or optionally substitutedcarbocycloalkyl, heterocyclo or optionally substituted heterocyclo,heteroaryl or optionally substituted heteroaryl, halo, aryl, aralkyl,(heterocyclo)alkyl, (heteroaryl)alkyl, alkoxycarbonyl, alkylcarbonyloxy,alkoxyalkanoyl, carboxyalkyl, amino or substituted amino, amido orsubstituted amido, and alkanoylamido, and A′ is selected from the groupconsisting of alkyl, alkoxy, alkoxyalkyl, aryloxy, cycloalkoxy,alkylthio, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkanoyl, aroyl,aminocarbonyl, aminoalkanoyl or optionally substituted aminoalkanoyl,carbocycloalkyl or optionally substituted carbocycloalkyl, heterocycloor optionally substituted heterocyclo, heteroaryl or optionallysubstituted heteroaryl, halo, aryl, aralkyl, (heterocyclo)alkyl,(heteroaryl)alkyl, alkoxycarbonyl, alkylcarbonyloxy, alkoxyalkanoyl,carboxyalkyl, amino or substituted amino, amido or substituted amido,and alkanoylamido, and X₁, X₂, and X₃ are independently selected fromthe group consisting of hydrogen, halogen, organosulfonyloxy,bromobenzenesulfonyloxy, methanesulfonyloxy,trifluoromethanesulfonyloxy, acyloxy, aryloxy, imidazolyl, —O—N═O, —NO₂,—OSO₃—, and —OPO₂(OH)⁻, with the proviso that X₁, X₂, and X₃ are not allhydrogen.
 27. The pharmaceutical formulation of claim 26, wherein Z isO.
 28. The pharmaceutical formulation of claim 26, wherein Z is S. 29.The pharmaceutical formulation of claim 26, wherein Y is O; X₁ and X₂are hydrogen; and X₃ is a halogen.
 30. The pharmaceutical formulation ofclaim 26, wherein A and A′ are independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, and aralkyl. 31.The pharmaceutical formulation of claim 30, wherein at least one of Aand A′ is a branched alkenyl group
 32. The pharmaceutical formulation ofclaim 31, wherein at least one of A and A′ is farnesyl orgeranylgeranyl.
 33. A compound having a formula selected from the groupconsisting of:

wherein n is from 0 to about 10, or a pharmaceutically acceptable salt,prodrug, or ester thereof.